人表皮细胞去分化的实验研究
发布时间：2018-08-18 16:01
【摘要】： 皮肤是人体最大的器官，位于体表，易于受伤，在创伤愈合过程中，表皮细胞进行增殖、游走、覆盖创面、分化从而完成创面的修复。表皮细胞在创面修复过程中发生一系列的表型变化，从而完成创伤修复。研究中发现表皮干细胞在维持表皮的自我更新，保持皮肤正常的表皮结构与功能方面起着重要作用。如何调动机体内的表皮干细胞来促进创面的愈合以及皮肤的功能性修复是人们普遍关注的研究热点。研究发现激活组织干细胞或通过诱导损伤附近细胞去分化，再次获得干细胞特性以诱导内源性再生能力是生物界普遍存在的现象。去分化的细胞重新获得干细胞形态和细胞分裂潜能，形成新的单元来修复损伤。近年来，在创伤修复领域里的去分化研究成为研究热点。已有研究发现在特殊条件下皮肤创面修复过程中表皮细胞可以发生去分化，从而促进创面的修复。 目前，研究表皮细胞的方法主要有：(1)人体的皮肤组织活体取材；(2)对培养的表皮细胞进行科学研究；(3)对动物皮肤及表皮细胞进行研究，从而推测人表皮细胞在各种干预因素作用下发生的变化及其机制。 本研究首先筛选一种合适的人表皮在体研究的动物模型，然后利用此动物模型对移植后的人表皮进行形态学研究。在此基础上，进一步设计去表皮基底层细胞的表皮移植的动物模型，从而对移植的表皮细胞去分化现象进行研究，为皮肤创伤修复研究提供理论基础。 1、动物模型的建立 应用中性蛋白酶消化后机械分离获得包皮表皮皮片，再把表皮皮片移植到裸鼠体内。设计两种动物实验模型：(1)在裸鼠背部创面游离移植表皮皮片；(2)在裸鼠背部皮下移植表皮皮片。分别于移植后3d、5d、7d取材。对标本进HE染色和CK10、CK14、p63的免疫组织化学染色进行形态学研究。 2、研究表皮皮片移植到裸鼠皮下后表皮细胞的表型改变 把表皮皮片移植到裸鼠皮下后于移植后3d、5d、7d取材，对标本进行HE染色和CK10、CK14、CK19、β1整合素、PCNA、p63、c-Myc的免疫组织化学染色进行形态学研究。 3、设计去基底层表皮皮片移植的动物模型，对移植的去基底层表皮进行去分化的研究 用Ⅳ型胶原反复粘贴并冲洗表皮皮片，以去除包括表皮干细胞在内的表皮基底层细胞。用免疫组织化学方法从形态学角度来验证此方法去除基底层细胞的可行性。 把去基底层细胞的表皮皮片用DAPI标记后移植到裸鼠皮下，术后7d取材，对标本进行HE染色和CK10、CK14、CK19、β1整合素、PCNA、p63、的免疫组织化学染色进行移植后表皮细胞表型改变的形态学研究。以α6整合素和CD71分子为表皮干细胞标志物应用流式细胞仪法检测移植后存活表皮细胞的表型改变。 1、表皮皮片移植到裸鼠皮下后皮片成活，成活率较高，达60％。移植后表皮细胞增殖、生长、分化，与在体皮肤创伤后的表皮细胞的变化一致。CK14、p63在基底层及附基底层阳性表达。而移植表皮皮片到裸鼠背部创面的方法表皮成活不理想，裸鼠自身表皮逐渐生长至移植表皮下方，并最终达到创面愈合，移植表皮皮片脱落。 2、表皮皮片移植到裸鼠皮下后基底层细胞增殖，p63、PCNA和CK14在基底层和副基底层阳性表达。CK10阳性表达于棘层和颗粒层，表达强度弱于移植前。表皮干细胞的标志物CK19和β1整合素阳性细胞数量明显多于移植前表皮。c-Myc在成活的表皮基底层和副基底层细胞中阳性表达。实验中发现，在成活的表皮皮片的棘层出现与基底层和副基底层细胞表型一致的散在分布的细胞或细胞岛。 3、Ⅳ型胶原处理前后的表皮片行HE和免疫组织化学检测显示：处理前的表皮片可见明显的表皮角，而处理后的皮片表皮角消失，皮片的最下层无排列紧密的细胞存在。处理前的表皮片基底层的细胞CK14阳性，基底层以上CK10染色阳性，处理后CK14阴性而皮片全层CK10染色阳性。移植的去基底层表皮细胞的皮片呈间断成活，以移植皮片有成活表皮细胞为成活标准，皮片存活率为33.3％。去基底层移植后7d的表皮皮片细胞排列分层不明显，无整齐排列的基底层细胞，与未去基底层的表皮皮片移植7d的HE结果有明显差别。去基底层表皮移植7d取材标本CK19和β1整合素染色阳性细胞呈多层散在分布，表皮中PCNA阳性细胞大量出现，细胞排列散在，分层不明显，p63阳性细胞也大量出现在成活的表皮片各个层次中，有的成团样聚集，分布不均。流式细胞仪检测结果：去基底层表皮皮片α6~+CD71~-细胞占0.016％，α6~+CD71~+占0.021％；移植后7dα6~+CD71~-细胞占1.366％，α6~+CD71~+占3.528％，，移植前与移植后α6~+CD71~-和α6~+CD71~+细胞比例有明显差异(P＜0.05)。 1、将分离的表皮皮片移植到裸鼠皮下可以成功建立人表皮在体研究实验动物模型。这为研究人表皮细胞在皮肤创伤修复中的作用提供了研究平台。 2、移植成活的表皮细胞与人体皮肤创面愈合过程中表皮细胞发生的表型变化相似，基底层细胞增生活跃，CK14、p63、PCNA阳性细胞增多，多层分布。成活的表皮皮片中表皮干细胞标志物β1整合素和CK19阳性细胞增多，提示表皮皮片的异种移植促进了表皮干细胞的分裂、增殖。 3、移植的表皮棘层出现散在分布的岛状细胞团，免疫组化染色证实为短暂扩增细胞或表皮干细胞。在皮肤创伤修复过程中，一定条件下表皮细胞可能发生去分化，从而促进创伤修复。 4、将分离的表皮用Ⅳ型胶原反复粘连并冲洗来可以成功去除表皮基底层细胞。 5、在去表皮基底层表皮皮片移植到裸鼠的动物实验模型研究中进一步证实了表皮细胞去分化这一生物学现象存在的可靠性。皮肤创伤修复过程中除了有静止干细胞的激活外，在一定条件下终未分化的表皮细胞去分化也参与皮肤创伤的修复。
[Abstract]:Skin is the largest organ in the human body, located on the body surface, easy to be injured. In the process of wound healing, epidermal cells proliferate, migrate, cover the wound, and differentiate to complete the wound repair. Epidermal cells undergo a series of phenotypic changes in the process of wound repair, thus completing the wound repair. Self-renewal plays an important role in maintaining the normal structure and function of the epidermis. How to mobilize the epidermal stem cells in vivo to promote wound healing and functional repair of the skin is a hot research topic. It has been found that activating tissue stem cells or inducing the cells near the injury to dedifferentiate can be obtained again. Dedifferentiated cells regain the morphological and mitotic potential of stem cells and form new cells to repair injuries. In recent years, the study of dedifferentiation in the field of wound repair has become a hot topic. It has been found that skin wounds under special conditions are damaged. During the process of surface repair, epidermal cells can dedifferentiate, thus promoting wound repair.
At present, the main methods to study the epidermal cells are: (1) human skin tissue in vivo; (2) scientific research on the cultured epidermal cells; (3) animal skin and epidermal cells were studied to speculate the changes and mechanisms of human epidermal cells under various intervention factors.
In this study, we first selected a suitable animal model for in vivo study of human epidermis, and then used this animal model to study the morphology of human epidermis after transplantation. It provides a theoretical basis for wound healing research.
1, the establishment of animal models.
Two animal models were designed: (1) epidermal grafts were transplanted freely on the wounds of the back of the nude mice; (2) epidermal grafts were transplanted subcutaneously on the back of the nude mice. P63 immunohistochemical staining was used for morphological study.
2, we studied the phenotypic changes of epidermal cells after skin grafting in nude mice.
The epidermal skin grafts were transplanted into nude mice subcutaneously and taken 3, 5 and 7 days after transplantation. The specimens were stained with HE and immunohistochemical staining of CK10, CK14, CK19, beta 1 integrin, PCNA, p63 and c-Myc.
3. Designing an animal model of dermal skin graft to dedifferentiate the transplanted dermal skin.
The epidermal basal layer cells, including epidermal stem cells, were removed by repeated adherence of collagen type IV and rinsing of epidermal skin grafts.
DAPI-labeled epidermal grafts were transplanted subcutaneously into nude mice. The specimens were stained with HE and immunohistochemical staining with CK10, CK14, CK19, beta-1 integrin, PCNA, p63. The morphological changes of epidermal cell phenotype were studied after transplantation. The phenotypic changes of viable epidermal cells after transplantation were detected by flow cytometry.
1. The survival rate of epidermal skin graft in nude mice was 60%. The proliferation, growth and differentiation of epidermal cells were consistent with the changes of epidermal cells after skin trauma in vivo. The epidermis gradually grew below the grafted epidermis and eventually healed and the grafted epidermis flaked off.
2. After subcutaneous skin grafting, the basal layer cells proliferated and p63, PCNA and CK14 were positively expressed in the basal layer and parabasal layer. CK10 was positively expressed in the spinous layer and granular layer, and the expression intensity was weaker than that before transplantation. Positive expression was found in the cells of the basal and parabasal layers. In the surviving epidermis, scattered cells or cell islands were found in the spinous layer, which was consistent with the phenotype of the cells in the basal and parabasal layers.
The results of HE and immunohistochemistry showed that the corners of the epidermis before and after treatment disappeared, and there were no close-packed cells in the bottom layer of the skin. The survival rate of the grafted skin was 33.3%. On the 7th day after the basal layer was removed, the cells in the epidermis were not obviously stratified, and the cells in the basal layer were not well arranged. After 7 days of epidermal grafting, the positive cells of CK19 and beta 1 integrin staining were scattered in many layers. PCNA positive cells were found in a large number of epidermis, the cells were scattered in many layers, and the layers were not obvious. P63 positive cells also appeared in many layers of the surviving epidermal grafts, and some of them were clustered. Flow cytometry showed that the percentage of alpha 6 ~ + CD71 ~ - cells was 0.016%, that of alpha 6 ~ + CD71 ~ + cells was 0.021%, that of alpha 6 ~ + CD71 ~ - cells was 1.366% and that of alpha 6 ~ + CD71 ~ + cells was 3.528% on the 7th day after transplantation, and that of alpha 6 ~ + CD71 ~ - and alpha 6 ~ + CD71 ~ + cells were significantly different before and after transplantation (P < 0.05).
1. Human epidermis can be successfully transplanted into nude mice to establish an animal model in vivo, which provides a platform for the study of the role of human epidermal cells in skin wound healing.
2. The phenotypic changes of epidermal cells during wound healing were similar to those of human skin. The basal layer cells proliferated and the positive cells of CK14, p63 and PCNA increased. The epidermal stem cell markers beta 1 integrin and CK19 positive cells increased in the surviving epidermal skin grafts, suggesting that xenotransplantation of epidermal skin grafts was possible. It promotes the division and proliferation of epidermal stem cells.
3. Scattered islands of cells appeared in the transplanted epidermal spinous layer. Immunohistochemical staining confirmed the transient expansion of cells or epidermal stem cells.
4, we can successfully remove epidermal basal layer cells by separating the epidermis with repeated adhesion and washing with type IV collagen.
5. In addition to the activation of stationary stem cells, the dedifferentiation of undifferentiated epidermal cells is also involved in skin wound repair under certain conditions. Repair.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329
本文编号：2189968
[Abstract]:Skin is the largest organ in the human body, located on the body surface, easy to be injured. In the process of wound healing, epidermal cells proliferate, migrate, cover the wound, and differentiate to complete the wound repair. Epidermal cells undergo a series of phenotypic changes in the process of wound repair, thus completing the wound repair. Self-renewal plays an important role in maintaining the normal structure and function of the epidermis. How to mobilize the epidermal stem cells in vivo to promote wound healing and functional repair of the skin is a hot research topic. It has been found that activating tissue stem cells or inducing the cells near the injury to dedifferentiate can be obtained again. Dedifferentiated cells regain the morphological and mitotic potential of stem cells and form new cells to repair injuries. In recent years, the study of dedifferentiation in the field of wound repair has become a hot topic. It has been found that skin wounds under special conditions are damaged. During the process of surface repair, epidermal cells can dedifferentiate, thus promoting wound repair.
At present, the main methods to study the epidermal cells are: (1) human skin tissue in vivo; (2) scientific research on the cultured epidermal cells; (3) animal skin and epidermal cells were studied to speculate the changes and mechanisms of human epidermal cells under various intervention factors.
In this study, we first selected a suitable animal model for in vivo study of human epidermis, and then used this animal model to study the morphology of human epidermis after transplantation. It provides a theoretical basis for wound healing research.
1, the establishment of animal models.
Two animal models were designed: (1) epidermal grafts were transplanted freely on the wounds of the back of the nude mice; (2) epidermal grafts were transplanted subcutaneously on the back of the nude mice. P63 immunohistochemical staining was used for morphological study.
2, we studied the phenotypic changes of epidermal cells after skin grafting in nude mice.
The epidermal skin grafts were transplanted into nude mice subcutaneously and taken 3, 5 and 7 days after transplantation. The specimens were stained with HE and immunohistochemical staining of CK10, CK14, CK19, beta 1 integrin, PCNA, p63 and c-Myc.
3. Designing an animal model of dermal skin graft to dedifferentiate the transplanted dermal skin.
The epidermal basal layer cells, including epidermal stem cells, were removed by repeated adherence of collagen type IV and rinsing of epidermal skin grafts.
DAPI-labeled epidermal grafts were transplanted subcutaneously into nude mice. The specimens were stained with HE and immunohistochemical staining with CK10, CK14, CK19, beta-1 integrin, PCNA, p63. The morphological changes of epidermal cell phenotype were studied after transplantation. The phenotypic changes of viable epidermal cells after transplantation were detected by flow cytometry.
1. The survival rate of epidermal skin graft in nude mice was 60%. The proliferation, growth and differentiation of epidermal cells were consistent with the changes of epidermal cells after skin trauma in vivo. The epidermis gradually grew below the grafted epidermis and eventually healed and the grafted epidermis flaked off.
2. After subcutaneous skin grafting, the basal layer cells proliferated and p63, PCNA and CK14 were positively expressed in the basal layer and parabasal layer. CK10 was positively expressed in the spinous layer and granular layer, and the expression intensity was weaker than that before transplantation. Positive expression was found in the cells of the basal and parabasal layers. In the surviving epidermis, scattered cells or cell islands were found in the spinous layer, which was consistent with the phenotype of the cells in the basal and parabasal layers.
The results of HE and immunohistochemistry showed that the corners of the epidermis before and after treatment disappeared, and there were no close-packed cells in the bottom layer of the skin. The survival rate of the grafted skin was 33.3%. On the 7th day after the basal layer was removed, the cells in the epidermis were not obviously stratified, and the cells in the basal layer were not well arranged. After 7 days of epidermal grafting, the positive cells of CK19 and beta 1 integrin staining were scattered in many layers. PCNA positive cells were found in a large number of epidermis, the cells were scattered in many layers, and the layers were not obvious. P63 positive cells also appeared in many layers of the surviving epidermal grafts, and some of them were clustered. Flow cytometry showed that the percentage of alpha 6 ~ + CD71 ~ - cells was 0.016%, that of alpha 6 ~ + CD71 ~ + cells was 0.021%, that of alpha 6 ~ + CD71 ~ - cells was 1.366% and that of alpha 6 ~ + CD71 ~ + cells was 3.528% on the 7th day after transplantation, and that of alpha 6 ~ + CD71 ~ - and alpha 6 ~ + CD71 ~ + cells were significantly different before and after transplantation (P < 0.05).
1. Human epidermis can be successfully transplanted into nude mice to establish an animal model in vivo, which provides a platform for the study of the role of human epidermal cells in skin wound healing.
2. The phenotypic changes of epidermal cells during wound healing were similar to those of human skin. The basal layer cells proliferated and the positive cells of CK14, p63 and PCNA increased. The epidermal stem cell markers beta 1 integrin and CK19 positive cells increased in the surviving epidermal skin grafts, suggesting that xenotransplantation of epidermal skin grafts was possible. It promotes the division and proliferation of epidermal stem cells.
3. Scattered islands of cells appeared in the transplanted epidermal spinous layer. Immunohistochemical staining confirmed the transient expansion of cells or epidermal stem cells.
4, we can successfully remove epidermal basal layer cells by separating the epidermis with repeated adhesion and washing with type IV collagen.
5. In addition to the activation of stationary stem cells, the dedifferentiation of undifferentiated epidermal cells is also involved in skin wound repair under certain conditions. Repair.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329
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